Electron microscope

ABSTRACT

The present invention provides an analysis of displacement by calculating the phase variance image P′ (k, l) between Fourier transformed images of paired images S 1  (n, m) and S 2  (n, m) to determine the center of gravity of δ peak appearing on the invert Fourier transform image of the images. The present invention provides numerous advantages such as a precision of displacement analysis of a fraction of pixel to thereby allow to improve the precision of focal analysis, or reduced number of pixels required to achieve the same precision, evaluation of reliability of the analysis by using the δ peak intensity, influence of varying background reduced by using a phase variance component. The improved performance by the present invention allows any operator skilled or not to achieve a best focusing.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. application Ser. No. 09/571,976, filedMay 16, 2000, recently issued as U.S. Pat. No. 6,570,156, the subjectmatter of which is incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an apparatus for automaticallycompensating for the focus and the amount of displacement using anelectron microscope image.

2. Description of the Prior Art

The inventor of the present invention have searched the Prior Art withrespect to apparatuses for compensating for the focus and the amount ofdisplacement by determining whether or not to automatically compensatetherefor by using an electron microscope image, or methods forcompensating for the amount of displacement of a continuously movingspecimen stage. As the result of search, there have been found threerelevant topics. First, a paper entitled as “The correction of imagedrift for autotuning a TEM using phase spectrum” by Norihiko Ichise etal., disclosed in the proceedings of the 51st academic conference ofJapan Electron Microscope Association, May 1995, pp. 161, discloses amethod for analyzing and compensating for the influence of image driftby using the phase spectrum method for analyzing the focus, astigmatism,and shifted axis. However, this paper does not disclose anything aboutthe improvement of the precision of analysis by means of the computationof the gravity center of a peak, and determination by means ofcompensation values, as well as about the compensation of the focus anddrift of a continuously moving specimen stage. Second, the JapaneseUnexamined Patent Publication No. Hei 10-187993, discloses an apparatusfor analysis of displacement between images by using the phase varianceof Fourier transform images on two photos taken in different conditions.

However, this application does not disclose anything about the feedbackto an electron microscopy apparatus and the spirit of the art but onlythe measurement of shape and distance of an object from a mark attachedto the object. Third, the Japanese Unexamined Patent Publication No. Hei10-339607, discloses the detection of amount of displacement by imageprocessing of the parallax of electron microscopy images, and thefeedback of the result thereof to the electron microscopy apparatus.More specifically, the images do not move before and after the angle ofincidence of electron beam varies if a specimen is located just on thefocus plane, however, if the specimen is located out of focusing planethen the images move before and after the angle of incidence of electronbeam. The relationship between the displacement D and out-of-focus F isD=M α (F+Cs α²), where a designates to the deflection angle of incidentelectron beam, M to the magnification rate, Cs to the coefficient ofspherical aberration, therefore the out-of-focus F may be given if theparallax displacement D is determined. There is disclosed an apparatusfor compensating for the focusing of objective lens system by storing ona memory a pair of images before and after altering the incident angleto apply a cross-correlation method or least-squares estimation methodto analyze the displacement D to determine the amount of defocus F.However, the analysis method of displacement using the phase variance ofFourier transform images is not disclosed. In an apparatus forautomatically compensating for the focusing or the amount ofdisplacement by using electron microscopy images, the performance maydepend on the settings of the photographic condition of images,analyzing images, and feeding back the analysis results. However, theoptimization is not performed with respect to the object, precision andtime of compensation.

The performance of the apparatus for automatic focusing an electronmicroscope in accordance with the displacement D between electronmicroscopy images such as the focus analysis using parallax and the likemay depend largely on the analysis method of displacement D. Theanalysis methods of displacement used heretofore, such as for examplethe cross-correlation method, least-squares method and the like, werelimited in terms of the precision by size of pixels of the electron beamdetector. The length of a side of pixel of a CCD camera used for presentelectron microscopy imaging is approximately 25 microns. The amount ofdefocus F corresponding to a pixel may depend on the angle and magnitudeof incident electron beam, the variance of incident angle α may beapproximately at most 0.5° due to the limitation by the hole diameter ofobjective aperture, and the magnitude should be the actual observationmagnitude. For example, at a magnification of 5,000, and an incidentangle variance of 0.5°, the focusing distance corresponding to thedisplacement D of a pixel is approximately 0.6 microns. This value isless than the precision level of focus compensation by a skilledoperator. The improvement of performance of apparatus such as refiningthe images used by the displacement analysis in favor of improving theprecision in the focusing analysis may cause excessive time spent foranalysis and excessive cost of hardware, thus is not practical.

The conventional method of displacement analysis has no functionality ofnumerical verification on whether or not an analysis has been performedcorrectly, so that the operator had to guess the result by the eye.Otherwise the operator had to compensate for the focusing based on thusobtained analysis results to verify that the compensation was doneaccurately. Since the automatic compensator has not assurance enough tocorrectly perform any analysis, there may be a need for a functionalityof aborting compensation when the result of analysis is not highlyreliable.

Furthermore, in the analysis of displacement in the prior art theanalysis was almost impossible when the image was shadowed by theobjective aperture. This phenomenon may be encountered routinely in theTEM observation, malfunction thereby thus may cause problems inpractice.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an apparatus forautomatic compensation by determining whether or not the focusing or theamount of movement is to be automatically compensated for by usingelectron microscope images.

The present invention uses the analysis method for determining thedisplacement of electron microscope images as will be described below.

For a pair of images containing displacement, a deflector means fordeflecting the incident angle to first electron microscope specimen isused to obtain a pair of images, to perform a Fourier transform thereonto compute the phase variant images. The analysis images obtained byhaving an invert Fourier transform or Fourier transform applied on thephase variant images may contain δ peaks at the positions correspondingto the displacement. The analysis images may be assumed to contain onlythe δ peaks, so that any peaks other than δ may be treated as noisecomponent.

Therefore, the computation of the gravity center of δ peaks will resultin an accurate position of the peaks even when the positions of δ peakscontains fractional component. The intensity of δ peaks computed afternormalizing the intensity of analysis images may be used as acorrelation, which indicates a match between images.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification illustrate embodiments of the invention and,together with the description, serve to explain the objects, advantagesand principles of the invention.

In the drawings,

FIG. 1 shows a schematic block diagram of an automatic focusing methodusing parallax;

FIGS. 2A and 2B show schematic diagrams of screen display examples forsetting parameters, and displaying the analysis result of the focusing;

FIG. 3 shows a flow chart of TEM imaging processes;

FIG. 4 shows a schematic diagram illustrating the principal of focusingmethod using parallax;

FIG. 5 shows a schematic diagram illustrating the computation ofdisplacement;

FIG. 6 shows a schematic diagram of basic configuration of an electrondetector 17 for TEM;

FIGS. 7A to 7C show schematic diagrams illustrating the position andintensity of peaks in analysis images, in case of a high contrastspecimen, a low contrast specimen, and a specimen with smalldisplacement, respectively;

FIGS. 8A and 8B show flow charts illustrating the identification ofpeaks corresponding to the displacement from analysis images, in case ofa process using a mask at the origin and a process outputting two peaks,respectively;

FIG. 9 shows a flow chart illustrating the focusing;

FIG. 10 shows a flow chart illustrating the automatic analyzer using aTEM;

FIGS. 11A to 11D, respectively, show schematic diagrams illustrating theidentification of analyzing areas by determining the direction and theshape of a mesh 22, in case of a TEM image of the mesh 22 at a lowermagnification power, an image labeled and binary coded, the relationshipbetween the gravity center 24 of holes 23 and the direction of mesh 22,and identified holes 23 each of which is split into a plurality ofanalysis areas;

FIG. 12 shows a flow chart illustrating analysis of the direction andshape of a mesh;

FIGS. 13A and 13B show schematic diagrams illustrating determining thepresence and absence of specimen in a TEM image, in each case of a TEMimage and image intensity histogram thereof when a specimen is present,and when no specimen is present;

FIG. 14 shows a flow chart illustrating viral inspection in an analysisarea;

FIG. 15 shows a display screen used for setting the parameters anddisplaying the progress of analysis in an automatic analysis;

FIGS. 16A to 16D show display screen examples for displaying the resultsof automatic analysis;

FIGS. 17A and 17B show schematic diagrams illustrating determining thepresence and absence of specimen in a TEM image, in each case of a TEMimage and the distribution of high frequency component of the Fouriertransform image thereof when a specimen is present, and when no specimenis present;

FIGS. 18A to 18C show schematic diagrams illustrating the position ofobserving field, positional setting of image shift, positional settingof specimen stage in the course of elapsed time, when the analysis areais shifted by moving a specimen on the specimen stage 18, image shiftingby means of deflective coil 16 for condenser system, and image shiftingby means of both the movement of specimen and image shifting,respectively;

FIG. 19 shows an electron microscope in accordance with the presentinvention;

FIG. 20 shows a process of viral analysis using the TEM;

FIG. 21 shows a process of evaluation of semiconductors using the TEM;

FIG. 22 shows a process of trimming from an TEM image patterns subjectedto be inspected and of comparing one with another thereof;

FIG. 23 shows a sequence of analysis when shifting inspection field bymeans of the specimen stage 18 or deflector coil 16 for condensersystem;

FIG. 24 shows a schematic diagram illustrating the principal of themethod of focus analysis using the parallax, (a) when irradiating aspecimen with an electron beam in parallel at the focal position, (b)when irradiating diagonally a specimen with an electron beam at thefocal position, (c) when irradiating diagonally an electron beam to aspecimen at the position out of focus;

FIG. 25 shows a schematic diagram illustrating a process for automaticfocusing using the parallax;

FIG. 26 shows a schematic diagram illustrating a process for automaticfocusing the displacement of a specimen by the deflector coil 16 forcondenser system;

FIG. 27 shows a schematic diagram illustrating a process for improvingthe precision of displacement analysis of a specimen by using a zoomlens system;

FIG. 28 shows a schematic diagram illustrating the focal distributionaround the optical axis in an electron beam astigmatic lens; and

FIG. 29 shows a schematic diagram illustrating a process forcompensating for astigmatism in an electron microscope.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A detailed description of preferred embodiments embodying the presentinvention will now be given referring to the accompanying drawings.

[First Embodiment]

FIG. 19 shows an transmission electron microscope (referred to andabbreviated to as TEM hereinafter) used in the embodiments disclosedherein in accordance with the present invention.

The TEM comprises an electron gun 11 and electron gun control circuit11′, a condenser lens 12 and condenser lens control circuit 12′, adeflective coil for condenser system 13 and deflective coil controlcircuit for condenser system 13′, an objective lens 14 and objectivelens control circuit 14′, a projector lens 15 and projector lens controlcircuit 15′, a deflective coil for condenser system 16 and deflectivecoil control circuit for condenser system 16′, an electron detector 17and electron detector control circuit 17′, a specimen stage 18 andspecimen stage control circuit 18′, a computer with control software andimage processing software 19. Each of control circuits may receivecontrol commands sent from the control software in the computer 19,perform controls and return the return value to the computer. Theelectron detector 17 is a detector constituted of a plurality of pixelssuch as a CCD camera, which may transmit signals of obtained imagesthrough the cable for image transmission to the storage device of thecomputer 19 or to the displacement analysis processor using phasevariance of Fourier transform images 20. The displacement analysisprocessor using phase variance of Fourier transform images 20 isconnected to the computer with control software and image processingsoftware 19.

FIG. 3 shows a flow chart of TEM imaging. An acceleration voltage isapplied to the electron beam that is the first charged particle beamgenerated by the electron gun 11, then the deflective coil for condensersystem 13 as a deflector means is used for adjusting the deflection ofbeam such that the electron beam passes through the optical axis, toverify that the electron beam reaches to the electron detector 17. Inthis document ‘z’ axis is defined as an axis in parallel to the opticalaxis, x-y plane is defined as the plane normal to the optical axis.After adjusting the condenser lens 12, a specimen 21 is set into theTEM, and a TEM image at lower magnification rate is observed. Theobjective aperture is inserted to the optical axis in order to increasethe contrast of TEM image. By gradually increasing the magnification ofthe projector lens 15, an observation field is selected and the focusingis adjusted to take an image of a second charged particle beam that isan electron beam transmitted through the specimen by the electrondetector 17.

To the analysis of focusing in the above focusing step is applied afocusing analysis method using parallax. In this method a first TEMimage obtained by an electron beam emitted at first incident angle inalmost parallel to the optical axis, and a second TEM image obtained byan electron beam emitted at second incident angle descend to an angle αfrom the optical axis are used. As shown in FIG. 4, when the beam isfocused somewhere out of focus, there will be generated somedisplacement between the first TEM image and second TEM image. Thedefocus F is correlated with displacement D by parallax in therelationship given by D=M α (F+Cs α²) The magnification rate M and theangle α may be set by the operator. Since the coefficient of sphericalaberration Cs is intrinsic to a specific apparatus, the amount ofdefocusing F may be specified by determining the displacement D of theimage pair. The present invention is characterized in that an analysismethod based on the phase variant analysis of Fourier transform imagesis applied to the determination of displacement D. As shown in FIG. 1,first and second TEM images may be obtained by using the electrondetector 17, with the angle of incidence of electron beam with respectto the specimen being varied by using the deflective coil for condensersystem 13 mounted above the objective lens 14. The first and second TEMimages thus obtained will be sent to the displacement analysis processorusing phase variance of Fourier transform images 20, from which thedisplacement D will be further sent to the computer with controlsoftware and image processing software 19. The computer 19 will computethe amount of defocus F from the displacement D to determine the currentof objective lens I_(obj) required for adjusting the focusing, andfinally the focus of the objective lens 14 will be thereby compensatedfor.

FIG. 5 shows a schematic diagram illustrating the computation ofdisplacement using the phase component of Fourier transform. For a pairof images with a displacement D=(dx, dy), by assuming S1 (n, m)=S2(n+dx, m+dy) the two dimensional discrete Fourier transform of S1 (n, m)and S2 (n, m) will be S1′ (k, l), and S2′ (k, l).

In accordance with the formula F {S (n+dx, m+dy))=F }S (n, m)} exp(idxk+idyl) of the Fourier transform, S1′ (k, l)=S2′ (k, l) exp(idxk+idyl) maybe obtained. The displacement in S1′ (k, l) and S2′ (k,l) above may be expressed as the phase variance exp (idxk+idyl)=P′ (k,l). Since P′ (k, l) is a wave with the cycle (dx, dy), then in an imageP (n, m) which is subjected to invert Fourier transform of a phasevariant image P′ (k, l), a δ peak will be appeared at the location (dx,dy). If (dx, dy) have a fraction, for example (dx, dy)=(2.5, 2.5), thenthe intensity of δ peak will be distributed in a manner aliquot to (2,2), (2, 3), (3, 2), and (3, 3). Since it can be assumed that in theimage P (n, m) only the δ peak may be present, the computation of fourgravity centers of intensity of pixels allows the correct determinationof δ peak even if a fraction is present. The cross-correlation method,which is used in the Prior Art as an analysis method, uses |S1′| and|S2′| as analysis images to compute the displacement based on thelocation with the maximum value in the images. Since the analysis imagescontains, together with information with respect to the displacement,any image intensity, i.e., the information about amplitude, theprecision of analysis will not be improved by the computation of thegravity center. It should be noted here that, when the information onamplitude is not totally eliminated, if an image is computed with theamplitude component suppressed by performing log or √{square root over ()} on the amplitude component of S1′ (k, l)·S2′ (k, l)*=|S1′| |S2′| exp(idxk+idyl) and then with the invert Fourier transform applied, a δ peakwill be appeared at the position (dx, dy) of displacement vector, theanalysis of displacement may be performed based on the image. Also itshould be noted that a δ peak will be appeared at the position (−dx,−dy) if the phase variant image P′ (k, l) is Fourier transformed, sothat the analysis of displacement may be performed on the Fouriertransformed image of the phase variant image P′ (k, l). Furthermore, anyone of other orthogonal transformations may be used instead of Fouriertransform to compute an image with peak corresponding to thedisplacement.

The analysis of displacement may be allowed if common component ispresent sufficiently in S1 (n, m) and S2 (n, m) when the variance in S1(n, m) and S2 (n, m) includes not only the displacement but also thevariable noise component or background behaviors, or when the image isdeformed more or less due to the change of angle of incidence ofelectron beam. In such a case any peaks other than δ peak may be treatedas noises. When the peak intensity δ is computed after normalizing theintensity of the entire image P (n, m), the intensity will be weaken ifthe unmatched area in the pair of images is larger, in other words, ifthe noise increases. Since the peak intensity will be stronger if theimages in the pair match in a larger extent, whereas the intensity willbe weaker if the images match in a smaller extent, the operator mayidentify the signal noise ratio, namely the reliability of results ofanalysis by expressing the peak intensity as the correlation valueindicating the match between images in the pair. In addition,malfunction may be prevented by setting the lower threshold value of thecorrelation to cause the adjustment of objective lens not to beperformed in case in which computed correlation value is less than thelower threshold value.

The analysis of displacement as described above has further an advantagethat it is hardly affected by the variance in the background since ituses the phase component of images. In the Prior Art, image analysis maynot be allowed if there is any variance in the background due to forexample the distribution of intensity of irradiation current, while theanalysis of displacement in accordance with the present invention may beallowed in the same condition. Also, the image analysis may not beallowed in the conventional analysis methods if the image contains forexample the shadow of objective aperture, the analysis of displacementin accordance with the present invention may be allowed if the commonarea of the pair of images is sufficiently presented even when theshadow of objective aperture is contained in some extent. As it isanticipated that a user not skilled in the TEM operation may use theautomatic focusing apparatus, it may be important that the TEMauto-focus works even when the fine adjustment of TEM is somewhatincomplete.

In order to perform the analysis of displacement, TEM images may becaptured by the electron detector 17 such as a CCD camera. The signaldetected by the electron detector 17 is amplified by an amplifier, thenquantized to send to the computer 19 or the displacement analysisprocessor using phase variance of Fourier transform images 20. It shouldbe noted that it is important to appropriately set the gain and offsetof the amplifier, otherwise almost all characteristics contained in theimages will be eliminated in the course of quantization. The electrondetector 17 comprises a functionality of automatic adjustment of gainand offset of the detector amplifier by computing the mean intensityvalue and dispersion of images so as to settle to specified values.Because it is anticipated that the specified mean and dispersion may notbe obtained by the gain and offset used, the detector comprises anotherfunctionality which may warn the operator when the contrast adjustmentis not complete to ask for further adjustments such as redefining theviewing field or tuning of TEM itself.

FIG. 6 shows a schematic diagram of basic configuration of an electrondetector 17 for TEM. The electron detector 17 comprises a scintillator71, a photo coupler 72, and a CCD camera 73. The electrons emitted tothe scintillator 71 generate photons. Thus generated photons will passthrough the photo coupler 72, which is made of a plurality of bundledoptical fibers, to the CCD camera 73 with positional information beingheld. The CCD camera 73 is constituted of a two dimensional array of aplurality of pixels. The charge generated by the photons incoming to theCCD camera 73 will be stored in each pixel. The stored charge will beread out as the output signal from each pixel. The gain of each pixel,in other words the intensity of signal output delivered by one incidentelectron may be determined by the light emitting efficiency of thescintillator 71, transmission efficiency of the photo coupler 72, andthe quantization efficiency of the CCD camera 73. Because theseconstants are not uniform from one pixel to another, a fixed pattern ispreformed in the electron detector 17.

The image captured by the electron detector 17 with a fixed pattern maystore a first contrast corresponding to the specimen structure togetherwith a second contrast corresponding to the fixed pattern of theelectron detector 17. When applying the analysis of displacement to theimage captured by the electron detector 17 with a fixed pattern, thefirst contrast corresponding to the structure of specimen may movebetween paired images S1 (n, m) and S2 (n, m), while on the other handthe second contrast corresponding to the fixed pattern of the electrondetector 17 does not, so that in the analysis image P (n, m) a firstpeak caused by the specimen structure will be observed at the positionrelative to the displacement, while a second peak caused by the fixedpattern will be observed at the origin. A very low contrast image ofspecimen structure such as a TEM image may often have the intensity ofsecond peak caused by the fixed pattern larger than the intensity offirst peak caused by the specimen structure. The images to which thedisplacement analysis has been applied heretofore were high contrast,high sharpness images obtained by an optical apparatus, with the effectof fixed pattern almost neglected such that the maximum intensity peakin the analysis image P (n, m) was determined to be the result ofanalysis. However, as the TEM images have considerable effect of thefixed pattern, the second peak caused by the fixed pattern might bedetermined as the result of analysis if the conventional peak detectionmethod is applied in which the maximum intensity peak is the result ofanalysis, such that the analysis may often be complete withoutdisplacement detected.

A preprocess, such as normalizing of gains, for minimizing the effect offixed pattern, by subtracting images by a fixed pattern previouslycaptured, may be incorporated in the CCD camera controlling software.The fixed pattern used for the computation should be updated at apredetermined interval because the pattern is affected by aging. Inorder to minimize the influence of the fixed pattern the routinemaintenance is indispensable. The change of fixed pattern due to thedifference in capturing conditions such as the amount of irradiation ofelectron beam and the like is inevitable if the apparatus is routinelymaintained. Although using only the normalization of gain may reduce theinfluence of fixed pattern, it will be difficult to completely eliminateit. Images of low contrast specimen structure such as electronmicroscopy images may have the second peak intensity larger than thefirst peak intensity of specimen structure even if any image processingsuch as gain normalization is performed on the paired images S1 (n, m)and S2 (n, m).

It is necessary for the displacement analysis of TEM images to add astep of automatically determining the first peak caused by the specimenstructure from the analysis image having the first peak caused by thespecimen structure together with the second peak caused by the fixedpattern. There are two algorithms as described below for automatic peakdetection. Both algorithms make use of the fact that the second peakcaused by the fixed pattern is observed at the origin.

Since the second peak intensity caused by the fixed pattern appears atthe origin, first algorithm applies a masking of the peak at the originfor substituting the intensity at the origin with a zero or apredetermined value. However, since the first peak may appear at theorigin, the application of the exception at the origin is required to bedetermined. Now referring to a specific example shown in FIG. 7, theflow of peak detection will be described below. It is now assumed theposition D1 and intensity I1 of the first peak 31 caused by the specimenstructure, and the position D2 and intensity I2 of the second peak 32caused by the fixed pattern. Since D2=0, there are predicted cases of|D1|>0 AND I1>I2 (FIG. 7A), |D1|>0 AND I1<I2 (FIG. 7B), and |D1|˜=0(FIG. 7C). For each case the intensity I of the maximum intensity peak33 retrieved by normalizing the analysis image P (n, m) will be comparedwith the intensity I′ of the maximum intensity peak 34 retrieved bynormalization after exclusion of (masking of) the peak in the analysisimage P (n, m) at the origin. In case of FIG. 7A, if the intensity ofsecond peak is as small as it can be neglected, the intensity of themaximum intensity peak will be the same before and after exclusion ofthe peak at the origin, whereas if the intensity of second peak isstrong enough then the intensity allocated to the second peak 32 byexcluding the peak at the origin moves to the first peak 31 and noise35, resulting in I≦I′. In case of FIG. 7B, without exclusion of the peakat the origin, the intensity allocated to the first peak 31 and thesecond peak 32 will be consolidated to the first peak 31, resulting inI<I′. In case of FIG. 7C, with exclusion of the peak at the origin, thefirst peak 31 will be eliminated together with the second peak 32, thenonly the noise 35 will increase, resulting in I>I′. The first peak 31may be determined based on the comparison of the result of analysis withand without exception of the peak at origin. FIG. 8A shows the flowchart of the peak identification process. In this flow chart theintensities of the peak of maximum intensity with and without exceptionof the peak at the origin, namely the correlation values will becompared, and the result of analysis without the exception of the peakat the origin will be selected if the correlation value decreases by theexception of the peak at the origin, while on the other hand theanalysis result with the exception of the peak at the origin will beselected if either the correlation value increases or remains.

As an alternative, assuming that there are two peaks in the analysisimage P (n, m), the algorithm may be predefined such that the positionsand intensities of two peaks will be output. The lower limit of thecorrelation should be redefined because the correlation value of each ofpeaks becomes weaker if there are two peaks. FIG. 8B shows two flowcharts of peak identification processes. In cases of FIG. 7A and FIG. 7Bthe peaks larger than the lower limit of correlation value are the firstpeak 31 and the second peak 32, so that two peaks will be output. Since|D1|>|D2|=0, when selecting the peak of larger displacement from withinthe output peaks, the first peak will be selected. In case of FIG. 7Csince the first peak and second peak are overlapped, there is only onepeak in the P (n, m). If there is only one peak which is larger than thelower limit of correlation value, then that peak will be selected as theanalysis result.

With respect to the setting of tilt angle α, when determining the amountof defocus F based on the amount of displacement D using the parallax,the tilt angle α of electron beam incident to the specimen will be used,so that the tilt angle α should be set accurately. The measurement ofthe tilt angle α may be accomplished by using the diffracted image of acrystalline specimen having a known diffraction grid coefficient, suchas Au and Si. As the diffraction grid coefficient is already known, ifthe wavelength of the incident electron beam is determined the diffusedangle for each pixel in the diffraction image may be calculated. Theactually measured value of the tilt angle α of incident electron beammay be obtained by determining the displacement D α in the firstdiffraction image taken at the first incident angle with the seconddiffraction image taken at the second incident angle to compute theproduct of the diffused angle for each pixel with the displacement D a.The tilt angle α of incident electron beam is approximately proportionalto the current value IBT flew through the deflective coil for condensersystem 13, however, as shown in FIG. 19 the deflective coil forcondenser system 13 is mounted above the objective lens 14 so that thefield caused by the objective lens 14 may alter the angle incident tothe specimen. Therefore the compensator item should be introduced intothe formulation of the tilt angle α of incident electron beam, using asa parameter the exciting current value I_(obj) of the objective lens 14.For instance, the formula α=A*I_(BH)+B*I_(obj)*I_(BH) may be used, whereA and B are constants intrinsic to the apparatus.

With respect to the amplitude of the tilt angle α, the larger the tiltangle α is, the smaller the amount of defocus F corresponding to thedisplacement D of image, therefore the improvement of the precision ofanalysis of the amount of defocus F may be estimated. However, thedecrease of common area in the pair of images may invoke a malfunction.The correlation decreases if the common area decreases to the half ofentire image or less, and the reliability of analysis results therebyextremely degrades. Thus the displacement D caused by the parallax isrequired to be set to be less than the half of the length of a side ofCCD camera. When the estimated range of defocus is wider at the samemagnification rate, namely in case of coarse focusing, the tilt angle αshould be set to smaller, whereas when the estimated range of defocus isnarrower namely in case of fine focusing, then the tilt angle α shouldbe set to larger. For example, in case in which the estimated range ofdefocus is 20 microns, the length of a side of electron beam detector is2 centimeters, and magnification rate is 50,000, then the angle α needsto be not more than 0.5 degree.

It may be possible that the analysis is unavailable because the commonarea of a pair of images is small due to the amount of defocus F thatwas larger than that was estimated. In order to address such acircumstance, a flow process should be provided in which a lower limitof the peak intensity should be set and then the magnification rateshould be lowered if the computed peak intensity goes down below thelower threshold to increase the percentage of the common area tocompensate for the focus in advance to thereby decrease the amount ofdefocus F to restore to the original magnification rate to measureagain. As an alternative in order to increase the common area the tiltangle α may be decreased.

In the TEM observation the objective aperture to be inserted to theoptical axis are often used to enhance the imaging contrast. It ispossible that, if the direction of incidence of electron beam ischanged, the electron beam will be out of optical axis so that the beamwill not pass through the aperture. In order for the electron beam offirst incident angle as well as the electron beam of second incidentangle to pass through the aperture, the tilt angle α should be setsmaller than the diameter of opening aperture. For instance, for theopening up to 10 microns, the tilt angle α should be set to 0.5° orless.

Since the second TEM image are to be taken with the incident electronbeam slanted, if the tilt angle α of the incident electron beam isexcessively larger then the image will be distorted due to the influenceof the eccentric axis, the distortion will cause an extreme decrease ofthe common area with the first TEM image, resulting in that the analysiswill be unavailable. In such a case the tilt angle α should be set againto smaller value.

The magnification rate M also is required for the calculation of defocusF. In the conventional TEM there are approximately 5% of magnificationerror.

In addition, when an optic lens is installed at the photo coupler 72connecting the scintillator 71 to the CCD camera 73, the magnificationerror of optic lens also should be considered. Now considering theinfluence of such an error with respect to the error rate of focusinganalysis in case in which there is an amount of error of M (1+Δ), i.e.,Δ in the magnification rate. It is assumed that for example adisplacement D1 was measured. The pure amount of defocus F1 may be givenby D1/[M (1+Δ) α]⁻¹−Cs α², but the amount of defocus F1′ may be given byD1/[M α]⁻¹−Cs α². The error of analysis of defocus F caused by themagnification error then may be F1−F1′=−ΔD1/ (1+Δ)Mα. The focusing errorcaused by the magnification error may be proportional to the amount ofdisplacement D1. In other words, when the displacement D=0, the focusingerror caused by the magnification error will be the smallest. Then thefocus compensation toward Fs=−Cs α² where the displacement D=0 may beattempted. When attempting to set the focus as Fs after the amount ofdefocus F1′ has been determined, the focus will be (F1−F1′)+Fs. Thedisplacement D2 at this stage will be measured as D2=−ΔD1. Since themagnification error Δ of the TEM is approximately 5%, a few times offocus compensation may lead to the displacement D˜=0. In this manner, itcan be seen that the focus error caused by the magnification error willbe sufficiently small by means of such a process flow that the optimumfocus specified may be set after the objective lens has been tuned tothe displacement D=0. The process flow may alternatively be such thatthe focus is adjusted to D=M Cs α³, where F=0 in the vicinity of D=0,not at the displacement D=0. In this case the influence of magnificationerror will be decreased together with the influence of the second peakcaused by the fixed pattern. The functionality is added which may abortthe compensation for focusing in case in which the repetition ofcompensation for defocus F=0 is more than two and the amount of defocusFn determined at nth path is larger than the amount of defocus F_(n−1)determined at n−1th path. This allows the repetition of compensation tobe held to the minimal requirement.

By taking above into consideration, focusing will be performed inaccordance with the flow chart shown in FIG. 9. At the beginning, afield of view on which focus will be analyzed will be selected. Theselection of a field of view includes also the setting of magnificationrate of the observation and of objective aperture. Then the displayexamples shown in FIGS. 2A and 2B will be used to set the optimal focus,lower correlation threshold, tilt α, and repetition of compensation. Therecommended values i.e., the initial default values of the optimalfocus, lower correlation threshold, tilt angle α, and repetition ofcompensation are preset in the computer, which may be changed by theoperator when required. Although the optimum focus is usually set toF=0, there may be cases in which under focus observation may bepreferable depending on the specimens. The tilt angle α is set atdefault to 0.5°, the largest angle α available for passing the electronbeam through the objective aperture of diameter up to 10 microns,however, there may be cases in which the tilt angle should be set to asmaller value, because the image distortion caused by the change ofelectron beam incident angle may severely affect to a certain fields ofview. The lower correlation threshold also depends on the photographicconditions such as the number of pixels of the analysis image and thelike. In order to optimize the tilt angle α and the correlation value,it will be necessary to provide a mode in which focusing will beanalyzed but not compensated. In general the tilt angle α used routinelywill be in the range from 0.2 to 0.5°. The lower limit of the tilt angleα and the upper limit of precision tolerance may be determined by theperformance of the system. Only the measurement may be performed bysetting the repetition of compensation shown in the display screen ofFIG. 2 to 0, and clicking the button 93 for repeating focusing. If theoperator cannot recall the recommended initial values during setting ofthe parameters, then by clicking on the “initialize” button 92, allparameters will default to the initial values.

Once the parameters are set, a pair of images may be taken by using theelectron detector 17. The conventional focus detectors of TEM oscillatessinusoidally the angle of incident electron beam by using the deflectivecoil for condenser system 13 and the operator observes the vibration ofTEM image.

Since the TEM image continues to vibrate in the conventionalconfiguration of circuitry, capturing of images is not available. Inorder to capture images, a control system is required in which an imageis captured upon receiving a signal instructing the capture of first TEMimage, then the incident angle of electron beam is changed to secondangle upon receiving a signal indicating the image capture has beencompleted, and a second image is captured thereafter. From a pair ofimages captured by using such a control system, an analyzing image P (n,m) will be computed to identify the peak corresponding to thedisplacement.

If any peaks corresponding to the displacement cannot be identified, aflow is provided so as to abort the focusing operation and to estimatethe cause for instructing the operator what to do next. The possiblecauses which may fail the analysis of displacement may be caused by aproblem in the image, such as for example no specimen found in the fieldof view, and an image extremely out of focus, or a problem of decreaseof the common area between images in the pair such as the amount ofdisplacement D excessively large due to the inclination too large ofincident electron beam angle, and the distortion of images affected bythe change of incident electron beam angle. In order to determine thecause, a fourth TEM image will be captured by translating the image by aknown amount of distance in a direction by using the deflective coil forcondenser system 16 at the first angle of incident electron beam toanalyze the displacement between the first TEM image and the fourth TEMimage. If, as a result, displacement analysis is impossible between thefirst and fourth TEM images, then there is a problem with respect to theimages such as no specimen in the field of view, or the image extremelyout of focus, or the like. To resolve such a problem an instruction maybe issued so as to decrease the magnification to perform a preliminaryfocusing at lower magnification. At a lower magnification a vaster fieldof view may be obtained, resulting in that the chance of finding thespecimen in the field of view will increase. In addition at a lowermagnification the influence of blurred image caused by the defocus maybe decreased and the sharpness may be improved so that the analysis ofdisplacement will be enabled. If the displacement between the first TEMimage and fourth TEM image may be analyzable, the cause may be thedecreased common area between images in the pair, therefore aninstruction should be issued to reduce the amount of tilt angle α. Thesystem will display an error message on the display screen as shown inFIG. 2(b). Although the correlation rate will be displayed the amount ofdefocus F that could not be determined will not.

Once the peak corresponding to the displacement has been identified, thecurrent through the objective lens will be adjusted by determining thedisplacement D therefrom to calculate the amount of defocus F by meansof the relation D=Mα (F+Cs α²).

Since the relationship between the current of objective lens and thefocal distance may vary depending on the parameters of the projectorlens that means the observing magnification rate, a relational table ora relation describing the current of objective lens with the focal pointfor each observation magnification rate is stored in the computer. Thecurrent of objective lens will be adjusted by using the table orrelation to determine the current required for focusing at the desiredfocus. In case in which the number of repetition of focusing is set totwo or more, another pair of images will be taken to analyze the focusto readjust the current of objective lens.

The automatic focus compensator system in accordance with the presentinvention incorporates a displacement analysis processor using phasevariance of Fourier transform images 20, implemented by a digital signalprocessor (DSP), which may complete the analysis of displacement of animage of 256 by 256 pixels within 30 milliseconds, in comparison withthe same calculation for 2 seconds by a conventional applicationsoftware. One focus compensation cycle will be completed within onesecond, including the image capture by the electron detector 17, theadjustment of the deflective coil for condenser system 13 and theobjective lens 14, and the system can also perform an iterativefocusing. The iterative focusing starts by clicking the button to repeatfocusing, as shown in FIG. 2, and the iterative focusing stops byclicking the button to stop focusing. Alternatively by clicking thebutton to repeat focusing the iterative focusing starts, and thefocusing may be stopped by clicking the same button again. Alternativelyby double clicking the button to execute focusing 93 the iterativefocusing starts, and the focusing stops by clicking the button toexecute focusing 93 again. In the course of repeating focusing the focuswill be automatically readjusted to an optimal focus even when the fieldof view changes by moving the specimen stage. When the first TEM imageand the second TEM image are taken during moving the specimen stage, theprecision of focal analysis will be degrade because of introduction ofthe displacement D caused by the parallax along with the displacement Dsof moved specimen stage, however, the precision of focusing required forthe specimen observation may be obtained since the observation may bestarted by stopping the specimen stage when the desired field of viewhas been found. In case in which the degraded precision of compensationby the moving specimen stage is concerned, the focus may be analyzed bymeasuring the moving speed of the specimen stage from the displacementbetween the first TEM image captured for the nth focusing analysis andthe first TEM image captured for the n−1th focusing analysis to estimatethe displacement Ds caused by the moved specimen from the first TEMimage to the second TEM image in the nth measurement, and by subtractingthe displacement Ds caused by the moved specimen from the displacementD+Ds between the first TEM image and the second TEM image to extract thedisplacement D caused by the parallax.

In the present system a malfunction checking functionality is built inso as to hold the focus setting if the correlation value is less thanthe lower threshold. The TEM image in general has low S/N ratio, and theimage of low S/N ratio may potentially have a higher probability thatthe displacement analysis is not executable. If the displacementanalysis is unavailable because of the probability, then the analysis inthe next turn will have a higher probability of obtaining a correctresult. Therefore the upper limit of the number of errors will bepredefined in the focus analysis. If the number of times that thecorrelation falls below the lower threshold exceeds the upper limit, thesystem provides a functionality to determine whether there has been anaccidental event, such as the TEM image of the structure of specimen wasnot captured by the electron beam detector because the electron beam wasblocked by for example the aperture and the like, and to display anerror message on the screen.

During the iterative focusing, the first TEM image by the first incidentangle and the second TEM image by the second incident angle arealternately displayed on the display screen. When the image processingspeeds up, the alternate display may be perceived as a kind of flickerof the display, giving the operator an uncomfortable feeling or adifficulty to observe fine structures. Therefore the present systemprovides a configuration in which the TEM image captured with the firstincident angle will be displayed on the screen, whereas the TEM imagecaptured with the second incident angle will not be displayed. Anotherconfiguration may also be provided, in which the TEM image observed atthe first incident angle and the TEM image observed at the secondincident angle are separately displayed, so that the influence of imagedistortion caused by the axial displacement of the incident electronbeam may be confirmed when required.

[Second Embodiment]

FIG. 19 shows a fundamental arrangement of TEM used in an automaticanalyzer. The TEM is comprised of an electron gun 11 and electron guncontrol circuit 11′, a condenser lens 12 and condenser lens controlcircuit 12′, a deflective coil for condenser system 13 and deflectivecoil control circuit for condenser system 13′, an objective lens 14 andobjective lens control circuit 14′, a projector lens 15 and projectorlens control circuit 15′, a deflective coil for condenser system 16 anddeflective coil control circuit for condenser system 16′, an electrondetector 17 and electron detector control circuit 17′, a specimen stage18 and specimen stage control circuit 18′, and a computer with controlsoftware and image processing software 19. Each of control circuits mayreceive control commands sent from the control software in the computer19, perform controls and return the return value to the computer. Thereis provided an image shift function for translating the TEM image byusing the deflective coil for condenser system 13 and the deflectivecoil for condenser system 16. The electron detector 17 is a detectorconstituted of a plurality of pixels such as a CCD camera, which maytransmit signals of obtained images at a higher rate through the cablefor image transmission to the storage device of the computer 19 or tothe displacement analysis processor using phase variance of Fouriertransform images 20. The displacement analysis processor using phasevariance of Fourier transform images 20 is connected to the computerwith control software and image processing software 19, which furthercomprises a software for pattern inspection and measurement.

FIG. 10 shows a process flow of the automatic analyzer using the TEM inaccordance with the present invention. An acceleration voltage isapplied to the electron beam generated by the electron gun 11, then thedeflective coil for condenser system 13 is used for adjusting thedeflection of beam such that the electron beam passes through theoptical axis, to verify that the electron beam reaches to the electrondetector 17. In this document ‘z’ axis is defined as an axis in parallelto the optical axis, x-y plane is defined as the plane normal to theoptical axis. After adjusting the condenser lens 12, a specimen 21 isput into the specimen chamber. The specimen 21 is thinned (sliced) toallow the electron beam to pass through, and then is mounted on ametallic support member that is also called a ‘mesh’ 22 (FIG. 11(a)).The mesh 22 is placed in a specimen holder, which is in turn placed onthe specimen stage to be observed. Since the diameter of the mesh 22 isapproximately 3 millimeters, it is difficult to accurately specify thedirection and position thereof when placing on a specimen holder.Therefore in accordance with the process flow shown in FIG. 12 the imageof the mesh 22 observed at a lower magnification rate is recorded toanalyze the direction, position, and shape of the mesh 22. The electronsmay transmit only through the void region called ‘hole’ 23. At first,the captured image will be binary coded to determine the connectivecomponents to label each region (see FIG. 11B). Then the surface area ofeach of labeled regions will be calculated. Since the holes are almostconstant in size, the modal (most frequent) value among the surface areavalues may be defined as the surface area of a hole. More specifically,among the regions labeled as shown in FIG. 11B, the regions having asize in proximity of the modal are regions labeled as 4, 5, 7, 8, 9, 12and 13, where the shape of entire holes is depicted. To analyze thedirection of the mesh 22, the center of gravity 24 of the labeledregions in which the hole 23 is entirely depicted will be computed. Thena combination having the shortest distance between the center of gravityof a region with respect to another will be determined (FIG. 11C). Forinstance, the nearest centers of gravity from the center of gravity 24of the labeled region 4 are the centers of gravity 24 of the labeledregion 5 and of the labeled region 8. The direction connecting thecenter of gravity 24 of the labeled region 4 with the center of gravity24 of the labeled region 5 will be defined as the x direction, while thedirection connecting the center of gravity 24 of the labeled region 4with the center of gravity 24 of the labeled region 8 will be defined asthe y direction. Since the shape of the hole 23 is known, the height Hof a side of hole 23 may be given from the surface area of the hole 23.By subtracting the height H of a side of hole 23 from the span of theshortest line between the nearest centers of gravity, the margin M ofthe mesh 22 may be computed. Once such calculation is completed, animage display that the x-y direction of the mesh 22 matches with thehorizontal and vertical direction of the display will be displayed. Eachhole 23 of the mesh 22 will be numbered. The operator may confirm onthis display at this point that the labeling has been correctlyexecuted. By directing a number labeled to a hole 23, a hole 23containing a specimen 21, i.e., a hole 23 to be inspected may be definedso as not to inspect other holes to shorten the time of inspection.

The presence or the absence of specimen in a hole may be determinedautomatically by image processing. The automatic decision uses ahistogram or Fourier transform image of the image intensity in the hole.The decision using a Fourier transform image uses the percentage of highfrequency component in the Fourier transform image.

When no specimen is present in the analytic area, as shown in FIG. 17B,the Fourier transform image contains only low frequency components, eventhough there is a certain fluctuation in the image intensity dependingon the distribution of the current density of irradiated electron beam.If a specimen is present which contains a fine structure such as in caseof biological specimens, the percentage of high frequency in the Fouriertransform image will be increased (FIG. 17A). It may be determined thatthe specimen is present in the analytic area to be inspected in case inwhich the percentage of high frequency component with respect to the lowfrequency component exceeds a certain threshold. When determining basedon the histogram of image intensity, the presence or absence of specimenwill be determined in accordance with the number of peaks present in thehistogram of the intensity of image. If no specimen is present in thehole, there will be only one peak (see FIG. 13B) even though the imageintensity may fluctuate more or less depending on the distribution ofthe current density of irradiated electron beam. On the other hand ifthere is a specimen in the hole, then there will be a plurality of peakspresent in the histogram of image intensity containing the specimen inthe hole (see FIG. 13A). Accordingly it may be determined that thespecimen is present in the hole when there is a plurality of peaks inthe histogram of image intensity. It should be noted that the presenceor absence of specimen may be determined based on the half-width ofpeaks in case of specimens which have a contrast so low that the peaksmay be overlapped.

Prior to analyzing the direction and the shape of mesh 22, it may bedesirable to adjust the height of the specimen holder. There may be acase in which the specimen holder is placed outside the focusing rangethat the objective lens maybe adjust, because of inappropriateadjustment of the specimen stage 18. When the stage is appropriatelyplaced within the focusing range, the magnification rate and the likemay vary due to the variation of lens condition when the current flewthrough the objective lens is drastically varied, therefore it may bedesirable to keep the height of the specimen holder approximatelyconstant. In the focusing analyzer as will be described later, afunctionality is provided to automatically adjust the height of thespecimen holder by analyzing the height of the specimen holder anddriving the specimen stage control circuit 18′.

The specimen holder may be inserted slantly because of for exampleinappropriate setting of specimen stage 18. If the specimen holder isseriously inclined, the condition of observation depends on the positionof hole in the mesh 22. Therefore by selecting a plurality of points inthe mesh 22, the position of those points and the height of specimendetermined by the analysis of defocus will be recorded. The inclinationangle of the specimen holder may be given by the height of specimen ineach of positions, and the inclination of the specimen holder can beadjusted accordingly.

The specimen stage control circuit 18′ contains the automatic adjusterof the inclination of the specimen holder.

Then items of inspection will be set. Most of automatic inspection ofbiological specimen are often the research of the shape and number ofviruses. For each of viruses various items of inspection such aspreprocess of image and the geometrical characteristics to be searchedmay be configured and stored in the computer 19 as a macro program. Forinstance, now considering a case of measuring the number of sphericalviruses having diameter in the range of approximately 20 nm to 30 nm,dispersed in the specimen, and the diameter thereof. The items ofinformation required to be inspected are solely the number and diameterof viruses, then the TEM image of the specimen having viruses stronglystained should be captured by using the electron detector 17, then theimage thus captured should be binary coded to extract the stainedregions, i.e., viruses to analyze the geometrical characteristics tomeasure the diameter of viruses. To this end, the size of the area to beanalyzed will be selected. The number of pixels contained in an imageused for the viral inspection may be 512 by 512 pixels, and whenmeasuring the diameter of viruses within an error of approximately 10%,in order for the diameter of a virus to be about 10 pixels the suitablelength of a side of square of the analytic area will be preferably about1 micron. If the length of a side of hole is 30 microns, then one holewill be divided into 30 by 30 areas, or 31 by 31 areas with an overlapof about 30 nm between areas so as to prevent the count from beingdropped therebetween. Once the division of areas has been completed adisplay as shown in FIG. 11D will be displayed on the screen to indicatethe areas to be inspected.

Since every areas to be inspected do not contain a specimen, if thespecimen is not present in an area to be inspected, it will be bettermove to next area immediately for saving the time of inspection. Thesystem provides a functionality of determining the presence or absenceof the specimen in a specific area to be inspected, by using thehistogram of image intensity or the Fourier transformed image in thatarea. As have been described above, the specimen was prepared such thatonly viruses are strongly stained, the area containing the specimen maybe presumably different in the image intensity from the area containingno specimen. If there is the specimen present in the area to beinspected, as shown in FIG. 13A, the histogram of the image intensitywill have a plurality of peaks. On contrary if the specimen is notcontained in the area to be inspected as shown in FIG. 13B, then therewill be only one peak, though the image intensity may fluctuate inaccordance with the distribution of the current density of irradiatedelectron beam. As a result, the presence or absence of viruses may bedetermined in accordance with the number of peaks present in thehistogram of image intensity. In case of the decision using the Fouriertransformed image the decision will be made based on the percentage ofhigh frequency component in the Fourier transformed image. If thespecimen is not present in the area to be inspected, as shown in FIG.17B, there is only low frequency component in the Fourier transformedimage. On the other hand, if the specimen is present in the area to beinspected, which contains a fine structure such as in case of biologicalspecimen, the percentage of high frequency component in the Fouriertransformed image will be augmented (see FIG. 17A). It may be determinedthat a specimen is present in the analytic area in case in which thepercentage of high frequency component with respect to the low frequencycomponent exceeds a certain threshold.

In a hole of 30 microns square, the diagonal distance is 42 microns. Ifthe specimen holder is inclined by 1°, a defocus of 0.74 micron may beoccurred in the hole. The contrast of TEM images is susceptibly affectedby the defocus. If there is a defocus in the order of submicron, theimage will be blurred or the contrast will be varied. Viral inspectionhas to be always performed with the image taken at a constant andprecise focus. The focusing should be well compensated for in the orderof submicron prior to starting a viral inspection.

The focal analysis using the parallax is applied to the focal analysisin such a focus compensation. A first TEM image taken with the electronbeam incident from a first angle approximately in parallel to theoptical axis and a second TEM image taken with the electron beamincident from a second angle inclined by a tilt angle α from the opticalaxis are used. As shown in FIG. 4, if the focus is not to the point,there may be a certain displacement of image between the first TEM imageand the second TEM image. The defocus F and the displacement D caused bythe parallax are related in D=M α (F+Cs α²). The magnification M and thetilt angle α may be selected by the operator. The spherical aberrationcoefficient Cs is intrinsic to the apparatus, so that the defocus F maybe identified if the displacement D between images in the pair isdetermined. The conventional method of analysis of displacement usedheretofore such as cross-correlation method, least-squares method andthe like, could not obtain a sufficient precision of focal analysisbecause the analytic precision of the analysis method of displacementdid not reach to less than one pixel. The present invention ischaracterized in that it applies a method of analysis based on the phasevariance analysis of the Fourier transformed image to the analysis ofthe displacement D. As shown in FIG. 1, the first and second TEM imageshaving the incident angle of the electron beam varied with respect tothe specimen by using the deflective coil for condenser system 13mounted above the objective lens 14 will be captured by the electrondetector 17. Thus captured first and second TEM images will betransmitted to the displacement analysis processor using phase varianceof Fourier transform images 20 and in turn the displacement D that isthe analytic result will be transmitted to the computer 19, which willcompute the amount of defocus F from the displacement D to determine thecurrent of the objective lens I_(obj) required to adjust the focus tothe target point, and then compensate for the focusing of the objectivelens 14.

FIG. 5 shows a schematic diagram illustrating the displacement analysismethod applied to the present invention. Now assuming a pair of imagesS1 (n, m)=S2 (n+dx, m+dy) having a certain displacement D=(dx, dy), andthe two dimensional discrete Fourier transform of S1 (n, m) and S2 (n,m) to be S1′ (k, l), and S2′ (k, l). From the formula F {S (n+dx,m+dy)}=F {S (n, m)} exp (idxk+idyl) of the Fourier transform, S1′ (k,l)=S2′ (k, l) exp (idxk+idyl) may be obtained. The displacement in S1′(k, l) and S2′ (k, l) above may be expressed by the phase variance exp(idxk+idyl)=P′ (k, l). P′ (k, l) is a wave with the cycle (dx, dy), thenin an image P (n, m) which is subjected to invert Fourier transform of aphase variant image P′ (k, l), a δ peak will be appeared at the location(dx, dy) Since it can be assumed that in the image P (n, m) only the δpeak may be present, the position of δ peak may be given by thecomputation of the center of gravity of the intensity of δ peak even ifa fraction is present.

The intensity of δ peak calculated after normalizing the intensity ofentire image P (n, m) will be weaker if the noise i.e., discrepancybetween images in the pair is increased. Thus the operator may identifythe signal-noise ratio, i.e., reliability of the analysis result byindicating the peak intensity as the correlation value. In the automaticcompensator the analysis is not always assured to be accurate in everyarea. Therefore, the lower threshold of the correlation will bepredetermined so as no to adjust the objective lens if the correlationvalue calculated is below the lower threshold to record the address ofthe analysis area along with the correlation value. For example, if theposition of mesh is shifted out of the point by an incorrect operationin the course of transfer of the specimen stage, more than half of acaptured TEM image will be occupied by the mesh 22 as well as the commonarea of the pair of images will decrease so that a sequence ofunanalyzable area will be left at the edge of holes 23. Once thedisplacement D has been analyzed, after the automatic inspectioncompleted, to allow to determine, based on the distribution ofunanalyzable area, whether the mesh was moved into the analysis area dueto the inaccurate operation of the specimen stage and at which step inthe course the incorrect transfer of the specimen stage was occurred,the operator may instruct to compute the focus F using the relation D=Mα (F+Cs α²) to determine the current value required for bringing to thespecified optimal focus to adjust the current of the objective lens.After the objective lens is adjusted, by performing another focusanalysis using the parallax to record the correlation value in thisdisplacement analysis and the current of the objective lens along withthe address of the analysis area, the status of the inspection may berecorded in greater details. The distribution of the specimen height maybe derived from the current of the objective lens set at the optimumfocus. In addition the image quality such as sharpness may be allowed tocompare by using the correlation value calculated at the samedisplacement D.

Once the focusing adjustment has been completed, a viral inspection maybe started in accordance with the process flow shown in FIG. 14.Although another TEM image may be taken for the inspection, a TEM imagewhich has been already taken at the incident angle 1 of the electronbeam with the optimum focus is recorded, and this image may be used forthe viral inspection in order to save the time of imaging. In a viralinspection, the TEM images will be binary coded to make the connectingcomponent to label every regions. Then, the surface area of each oflabeled regions will be computed to eliminate the region having lessthan a predetermined surface area, because these smaller regions may bedetermined as noise. Then, the characteristic amount of biologicalspecimen will be computed from the roundness and moment of each of thelabeled regions, and the region identified to be closer to a roundnesswill be determined to be likely a virus, thereby the diameter(biological information) will be derived from the surface area. Thenumber of viruses and the diameter of each virus will be recordedtogether with the address of the analytic area in a similar manner.

Once the inspection has been completed in one area to be analyzed, thespecimen stage 18 may be used to transfer the specimen to move to a nextarea to be inspected to start the inspection. The precision of fineadjustment of the specimen stage 18 may be described by the registeringaccuracy and the back-rush thereof. The registering accuracy is theaccuracy of transfer in a constant direction of moving the specimenstage, the back-rush is the distance of slipping at the time of turningthe direction. In the products currently available in the market, theregistering accuracy is achieved in the order of about 1.2 nm, back-rushin the order of about 0.02 micron. In case of analytic areas of diameterof 30 microns, the specimen transfer may be accomplished by using thespecimen stage 18.

However, when moving the specimen stage 18, a certain amount of specimendrift may be occurred by the inertia of the specimen stage 18. In thefocusing analysis using the parallax, the precision of focusing analysiswill be degraded if the displacement D caused by the parallax isintermixed with a certain amount of displacement Ds caused by the driftof specimen. To avoid this, a third TEM image, which may be taken at thefirst incident angle of the electron beam at a second time differentfrom the time at which the first TEM has been taken will be used. Theamount of specimen drift may be computed from the amount of displacementbetween the first TEM image and the third TEM image. This displacementanalysis also may be performed by means of the displacement analysisusing phase variance of Fourier transform images. The precision of focusanalysis is likely to be degraded unless the displacement Ds caused bythe specimen drift is analyzed at the same analytic precision as theanalysis of displacement D. In addition, the measurement of drift ofspecimen has to be completed within a very short period of time, and theamount of displacement caused by the specimen drift are to besignificantly small. The conventional analysis of displacement, in whichthe analytic precision may be limited by the size of a pixel, obviouslyhas not sufficient precision. The displacement D caused by the parallaxmay be given by subtracting the displacement Ds caused by the specimendrift from the amount of displacement between the first and second TEMimages. In addition, the blur caused by drift in the captured images maybe removed by performing an automatic drift compensation for operatingthe deflective coil for condenser system 16 so as to cancel thedisplacement Ds caused by the specimen drift.

The occurrence of specimen drift which may affect to the focal analysismay be estimated in some extent, such as at the time when the electronmicroscope has been just powered on, during the period of time until thedifference of temperature in the microscopy and/or the electron gun willbe settled, and at the time immediately after the moving specimen stage18 is stopped. Since the efficiency of analysis will be lowered if anumber of images are to be captured, then the condition of observationto capture the third TEM image for compensating for the drift may bepredetermined, and when the condition matches, the third TEM image willbe taken along with the first and second TEM images so as to enable toeliminate the influence of drift. If the algorithm is implemented inwhich only the first and second TEM images are captured once the driftdecreases, in other words when the amount of displacement between thefirst TEM image and the third TEM image reaches to or in the vicinity ofzero, the accurate compensation of focusing may be accomplished with theleast number of TEM images required.

The efficiency of inspection will be degraded if a third TEM image usedfor compensating for the drift of specimen is taken each time thespecimen stage 18 moves. Therefore, the transfer of the specimen stage18 may be limited to the transfer between holes and the transfer betweenthe analytic areas may be performed by the deflective coil for condensersystem 16. The number of third TEM image to be taken will besignificantly decreased since the compensation of specimen drift causedby the inertia of the stage transfer will be performed only when movingbetween holes. Other examples requiring the transfer of analytic area byusing the deflective coil for condenser system 16 include, for example,a case in which the final precision is insufficient with the precisionof fine focus adjustment of the specimen stage 18 with respect to thetransfer of analytic area because the analytic areas are subdivided intosmall areas.

If the size of a hole is enough vast so that the deflective coil forcondenser system 16 cannot follow, the image may be shifted by movingthe specimen stage 18 into a direction at a constant velocity and usingthe deflective coil for condenser system 16 to move at an approximatelysame velocity. FIG. 18 C shows the location of the field of view whenthe transfer of the specimen stage is used together with the imageshifting in order to move between analytic areas, the parameters of thedeflective coil for condenser system 16, and the position of thespecimen stage 18 along with the elapsed time. Here Tc designates to thetime required for taking a TEM image, Ts to the time required fortransfer of the field of view by the specimen stage, and Ti to the timerequired for the transfer of the field of view by the deflective coilfor condenser system 16. The transfer of the field of view by using thespecimen stage 18 may be accelerated but the speed-up is limited due tothe influence of inertia when moving the stage and the back-rush, thetransfer of field of view by using the specimen stage 18 may not beaccelerated faster than the transfer of field of view by using thedeflective coil for condenser system 16. If the field of view istransferred solely by means of the specimen stage 18, the time ofinspection will be prolonged as shown in FIG. 18A. On the other hand thedeflective coil for condenser system 16 has a disadvantage of narrowerrange of transfer. As shown in FIG. 18B, when the transfer is out ofrange of the deflective coil for condenser system 16 the specimen stage18 should be used to move the specimen. Accordingly, when moving thespecimen stage 18 at a constant velocity as shown in FIG. 18C whileusing the image shifting by means of the deflective coil for condensersystem 16 so as to cancel out the movement of the specimen stage, stillimages of each of analytic areas may be taken even when the specimenstage 18 is in the course of transfer. In this manner the transfer ofthe field of view in a vast range may be carried out at high speedwithout influence of the backrush and inertia of transfer of thespecimen stage 18.

The analysis may be performed in a vaster range if the position of thedeflective coil for condenser system 16 is approximately constant at themoment of the beginning of inspection for each analysis area (see FIG.18). To do this, the transfer velocity of the specimen stage 18 shouldbe set, by calculating the time window T required for both thecompensation and inspection carried out in each analysis area such thatthe transfer to the next analysis area by means of the specimen stage 18may be completed before this time window T expires. The amount of imageshifting by means of the deflective coil for condenser system 16 will bemanaged so as to be able to cancel thus decided transfer speed of thespecimen stage 18. In order to match the transfer speed of specimen bymeans of the specimen stage 18 with the shift speed of the deflectivecoil for condenser system 16, the analysis of displacement in the fieldof view may be performed by the displacement analysis using phasevariance of Fourier transform images. As shown in FIG. 26, after settinga first time and a second time, a first TEM image at the first time aswell as a third TEM image at the second time will be captured by usingthe electron detector 17. Thus captured first and second TEM images willbe transmitted to the displacement analysis processor using phasevariance of Fourier transform images 20, from which the displacement Dresulted by the analysis will be further sent to the computer withcontrol software and image processing software 19. The computer 19 willcompute the moving velocity of the field of view based on thedisplacement D to determine the parameters of deflective coil forcondenser system 16, which are required for the transfer speed of thefield of view to be zero, and to adjust the deflective coil forcondenser system 16 based on the parameters thus determined.

It is preferable to keep constant the time T of inspection for eachanalysis area, since the positional precision of the specimen stage 18is higher when the transfer speed of the specimen stage 18 is constant.Thus it is preferable to keep constant the number of images to be takenfor each analysis area. Otherwise if a third TEM image for analyzing thedisplacement of the analysis area is taken for each of analysis areas,the precision of displacement compensation and the precision of thefocusing analysis may be improved, whereas the efficiency of inspectiondeteriorates. Accordingly, The transfer of the field of view by usingthe specimen stage 18 as well as the transfer by using the deflectivecoil for condenser system 16 will be adjusted in the stage of adjustingthe microscope prior to the viral inspection. Alternatively any analyticareas inappropriate for the viral inspection may be predefined so as notto perform focusing in the areas, but the first TEM image and third TEMimage may be taken to adjust the transfer of the field of view. Byassuming that the field of view in the usual analysis areas will bevirtually stationary, the first TEM image and second TEM image will betaken for adjusting the focus.

The items displayed in the course of inspection may be selected by theoperator as required. For example, items as shown in FIG. 15 may bedisplayed on the screen. On the screen, the image of the analysis areasinto which the image of the mesh taken 22 are divided will be displayedin a window, in which the analysis area that the inspection has beencompleted, area that the inspection is in progress, and area that is notyet inspected will be displayed in different colors respectively, inorder for the operator to be able to make use thereof for theunderstanding of the progress of the inspection and for the estimationof the time of completion. There may be provided also a window showing atable 94 for sequentially displaying the result of analysis in each ofthe analysis area, and a histogram 95 for displaying the cumulativevalues of the results. Also a window for displaying the result of focalanalysis may be provided, which may display the height of specimencalculated from the correlation value between the paired images and fromthe result of focal analysis. For the reference point of height ofspecimen an appropriate location in the specimen may be selected beforeor after inspection and that location may be specified by clicking thereset button 96. Within a window of TEM image for use of viralinspection a layer is provided for displaying circles 97 that indicatesthe position and the size of viruses identified. The operator who checksthe results of viral inspection, focusing, and TEM image can abort thatinspection if something goes wrong. The items specified by the operationamong the result of viral inspection, focusing, and TEM image will bestored in the memory so as to allow the operator to display, after theinspection, the result of inspection of the analytic areas in whichanomalies is prospected to be happened, based on the information such asthe correlation value and the height of specimen and to confirm theinspection status.

The mesh 22 is also used for the display of analytic result after theinspection. The inspected holes are divided into several areas, and theresult with respect to each area may be displayed as black and white orin full color in accordance with the selection of displayed items. Forinstance, when the operator selects the number of viruses for thedisplay item, each area will be colored in accordance with the number ofviruses, as shown in FIG. 16B. The TEM image taken for each of analysisareas may be shown on the screen by for example specifying the number ofarea in interest, or by double clicking on the spot of the analysisarea. Among items stored in the memory, only the items specified by theoperator will be displayed on the screen. For example, the result ofviral inspection in that area in interest, the height of specimen, thecorrelation value may be displayed along with the TEM image display(FIG. 16A).

The distribution chart of the height of specimen and the correlation maybe used for outlining the inspection status and for evaluating thereliability of the inspection result. Since biological specimens are ingeneral sliced into thin sections of thickness of about tenthnanometers, which may be considered to be almost flat. The change of theheight of specimen may be caused by the curved or inclined mesh thatmounts the specimen. When plotting the distribution of the height ofspecimen the distribution of height should form a curved surface of thekind relatively smooth. If the height of specimen changes abruptly, thenit can be concluded that either an incorrect operation occurred in thefocusing analysis of that area, or the sectioned specimen was blown upfor some reason. In either case, the reliability of the result ofinspection of the analysis area in question is not sufficient. Forexample, when considering the distribution of the height of specimen asshown in FIG. 16C, as the height of specimen in the region 25 isdifferent from other regions, it can be concluded that the reliabilityof the result of inspection in the section 25 is not eligible. In orderto remove the result of analysis in this region, the setting may beconfigured so as no to use the result of analysis in any analytic areaout of specified range of the height of specimen. This means that thedistribution of the height of specimen may be used as a sort of filterapplied to the result of analysis. Alternatively, instead of configuringa filter based on the height of specimen, the curvature obtained fromthe distribution of height may be used for configuring a filter. When adistribution of correlation as shown in FIG. 16D has been obtained, thiscorrelation may be also used for the evaluation of the sharpness of TEMimages. In case of biological specimens, if the section of slicedspecimen is thick, even images observed at the optimum focus will beblurred. With blurred images the correlation will be degrades and theerror encountered at the time of binary coding will be larger so thatthe precision of measurement of the diameter of viruses will be lowered.By using the distribution of correlation as a filter, in a similarmanner to the distribution of the height of specimen, the analysisresult in the region 26 having a lower correlation than others may beeliminated. By estimating the measurement error of the viral diameterbased on the correlation values, the measurement error may be used as aweighting function when creating a distribution chart of the diameter ofviruses.

[Third Embodiment]

FIG. 19 shows a fundamental arrangement of a transmission electronmicroscope (TEM) for use in an embodiment in accordance with the presentinvention. The TEM is comprised of an electron gun 11 and electron guncontrol circuit 11′, a condenser lens 12 and condenser lens controlcircuit 12′, a deflective coil for condenser system 13 and deflectivecoil control circuit for condenser system 13′, an objective lens 14 andobjective lens control circuit 14′, a projector lens 15 and projectorlens control circuit 15′, a deflective coil for condenser system 16 anddeflective coil control circuit for condenser system 16′, an electrondetector 17 and electron detector control circuit 17′, a specimen stage18 and specimen stage control circuit 18′, and a computer with controlsoftware and image processing software 19. Each of control circuits mayreceive control commands sent from the control software in the computer19, perform controls and return the return value to the computer. Theelectron detector 17 is a detector constituted of a plurality of pixelssuch as a CCD camera, which may transmit signals of obtained imagesthrough the cable for image transmission to the storage device of thecomputer 19 or to the displacement analysis processor using phasevariance of Fourier transform images 20. The displacement analysisprocessor using phase variance of Fourier transform images 20 isconnected to the computer with control software and image processingsoftware 19.

FIG. 3 shows a flow chart of TEM imaging. An acceleration voltage isapplied to the electron beam that is the first charged particle beamgenerated by the electron gun 11, then the deflective coil for condensersystem 13 as a deflector means is used for adjusting the deflection ofbeam such that the electron beam passes through the optical axis, toverify that the electron beam reaches to the electron detector 17. Afteradjusting the condenser lens 12, a specimen 21 is set into the TEM, anda TEM image at lower magnification rate is observed. The objectiveaperture is inserted to the optical axis in order to increase thecontrast of TEM image. By gradually increasing the magnification of theprojector lens 15, an observation field is selected and the focusing isadjusted to take TEM images as required.

To the analysis of focusing in the above focusing step is applied afocusing analysis method using parallax. In this method a first TEMimage obtained by an electron beam emitted at first incident angle inalmost parallel to the optical axis, and a second TEM image obtained byan electron beam emitted at second incident angle descend to an angle αfrom the optical axis are used. A functionality for deliberatelychanging the irradiating angle of the electron beam into the specimen byusing the deflective coil for condenser system 13 as shown in FIG. 19 iscalled a wobbler, which may be used for transform an amount of defocusinto a parallax. Now referring to FIG. 24, the principal of operation ofthe wobbler and the mechanism of occurrence of parallax will bedescribed below in greater details. In FIG. 24(a) there is shown anoptical geometry in case that a specimen is just placed at the focalplane (F=0), and the electron beam is emitted in parallel to the opticalaxis of the system (α=0). In the figure the electron beam is emitted tothe specimen (shown as an arrow) in a direction from the top toward thebottom of the drawing. Part of electron beam will be diffracted withinthe specimen. For instance in case of a crystalline specimen theelectron beam will be dispersed to a specific direction which maysatisfy the Bragg's rule, and the rest thereof will be transmittedthrough the specimen without changing the direction. The objective lensplaced below the specimen may have the characteristics similar to anyordinary optical convex lens, and act to collimate the electron beam.The electrons diffracted to the same direction will be converged to apoint below the lens; the converged electrons forms so-called thediffraction plane (back focal plane). Below the diffraction plane theelectrons diffracted and transmitted at an identical point will beconverged to form the TEM imaging plane. At the TEM focal plane, thesize of the specimen is magnified by M times, depending on theprojection magnification rate M of the objective lens. If F=0 and α=0,then the image of the arrow is correctly focused at the TEM image planeas shown in FIG. 24(a), without displacement from the optical axis(c.f., D=0). If otherwise the specimen is placed at the focus position(F=0) but the electron beam is tilted by an incident angle α, as shownin FIG. 24(b), by means of the wobbler, then the electron beam will besubjected to collimate to another focal position, displaced from theaxis of the objective lens. This may result in a displacement of fieldof view, D=Cs M α³, because of the influence of the spherical aberrationwhich is intrinsic to the convex lens system as similar to the opticallens, where Cs designates to a spherical aberration index which is anintrinsic value of a specific lens. If otherwise the specimen is not atthe focal position and the electron beam is tilted by an incident angleα by means of the wobbler, then the amount of displacement will beworsen as shown in FIG. 24(c). At the amount of defocus F, the positionof specimen will be shifted by an amount a F to the direction normal tothe optical axis the image at the TEM imaging plane will be magnified bythe magnification rate M of the lens to result in a parallax M α F. Thusthe total amount of parallax together with the displacement due to thespherical aberration will become an amount indicated by D=M α (F+Cs α²).It can be clearly seen from this equation that, the parallax D will bezero if α=0, irrelevant whether the specimen is place at the focalposition or not. By using the wobbler in such a manner as describedabove when photographing two pairs of images which have different α eachother, the amount of defocus F may be specifically identified based onthe amount of displacement D in the paired images. The parallax due tothe aberration (Cs M α³) is fewer than the parallax due to the defocus(M α F) in the order of one figure or less. Therefore by minimizing theparallax a focus compensation at higher precision may be achieved. Thefocusing using the wobbler may be considered to be completed if theparallax becomes less than Cs M α³.

The displacement D in the paired images may be given by using thedisplacement analysis using phase variance of Fourier transform images.

FIG. 5 shows a schematic diagram describing the displacement analysisusing phase variance of Fourier transform images. Assuming that a pairof images S1 (n, m)=S2 (n+dx, m+dy) has the displacement D=(dx, dy), andthe two dimensional discrete Fourier transform of S1 (n, m), and S2 (n,m) is S1′ (k, l), and S2′ (k, l). In accordance with an equation of theFourier transform, F {S (n+dx, m+dy)}=F {S (n, m) } exp (idxk+idyl), thepair of images may be expressed as S1′ (k, l)=S2′ (k, l) exp(idxk+idyl). The displacement D of S1′ (k, l), and S2′ (k, l) may beexpressed by a phase variance exp (idxk+idyl)=P′ (k, l). Since P′ (k, l)is a wave with the cycle (dx, dy), then in an image P (n, m) which issubjected to invert Fourier transform of a phase variant image P′ (k,l), a δ peak will be appeared at the location (dx, dy). Since it can beassumed that in the image P (n, m) only the δ peak may be present, thecomputation of the center of gravity of the intensity of δ peaks allowsthe correct determination of δ peak even if a fraction is present in theposition of δ peaks.

When the peak intensity δ is computed after normalizing the intensity ofthe entire image P (n, m), the intensity will be weaken if the unmatchedarea in the pair of images is larger, in other words, if the noiseincreases. By expressing the peak intensity as the correlation value thematch between images in the pair may be evaluated.

By using the apparatus as shown in FIG. 25, focusing will be carried outin accordance with the flow chart shown in FIG. 9. At the beginning, afield of view on which focus will be analyzed will be selected by thefine-tuning mechanism of the specimen provided by the specimen stage 18.The selection of a field of view includes also the setting ofmagnification rate of the observation and of objective aperture. Thenthe display shown in FIG. 2 will be used to set the optimal focus, lowercorrelation threshold, tilt angle α, and repetition of compensation.Once the parameters are set, a pair of images may be taken by using theelectron detector 17. From a pair of images captured, an analyzing imageP (n, m) will be derived to identify the peak corresponding to thedisplacement. Thus derived displacement D will be further sent to thecomputer with control software and image processing software 19. Thecomputer 19 will compute the focus F by means of the relation D=M α(F+Cs α²), and the current of the objective lens I_(obj) to be adjustedcorresponding to the focus F, then send this value to the objective lenscontrol circuit 14′ to carry out the adjustment of the current of theobjective lens.

In this embodiment, exemplary inspections of virus or semiconductormemory using an electron microscope implementing the focusingcompensator as have been described above will be described below. Sincean electron microscope has a capability of resolution in the level ofatoms, may obtain a range of contrasts in accordance with the structureof the specimen, a variety of observations is performed in biological aswell as non biological fields. In case of viral inspection, the viruses,such as AIDS and Influenza, which are too small to be identified by anoptical microscope, should be identified in shorter times, to determinewhether viruses is present or absent, and to diagnose whether theinfection is present or absent for a number of patients. In such asituation, heretofore, the operator of electron microscopy inserts thespecimen by hand to the electron microscope operating in manual mode toevaluate by naked eyes. The inspection of semiconductor memory isanother example. A specimen, which was picked up in an appropriatemanner and processed to the shape suitable for observation will be setto an electron microscope and observed. Since the density of integrationin recent years is increasingly becoming higher than ever and the numberof field of view to be observed is increasing more and more, it isnearly impossible for an inspector to manually find every defect. Inaddition, since most of specimens are not made flat, and are not alwaysplaced in a plane perpendicular to the electron beam, the focusing pointwill be gradually shifted when the field of view is continuouslytransferred one after another, so that the focusing should be done eachtime. Accordingly, the throughput of the observation by means ofautomatic control of the inspection process is being a matter of utmostconcern. An example of viral inspection in accordance with a preferredembodiment of the present invention will be described below in greaterdetails, with reference to FIG. 20, using the functionality of automaticfocus compensation in accordance with the present application. Similarlyto the first embodiment above, the displacement D will be calculatedfrom a pair of taken images to derive the amount of defocus F and thecurrent of the objective lens I_(obj) corresponding thereto. Based onthese parameters the objective lens will be immediately readjusted.Thereafter another image will be taken. Otherwise the image forinspection will be taken after repeating the fine adjustment of specimenand the focusing for several times until the target field of view willbe seen on the image. The comparison with the image registered for thereference with respect to the viruses to be extracted will be performedthereafter.

Also in this preferred embodiment, similar to the analysis of thedisplacement D, the consistency of the shapes will be evaluated by usingthe displacement analysis based on the phase variance of two Fouriertransform images to derive the correlation value to determine that thevirus is found therein when the correlation value is less than thepredetermined lower correlation threshold (limit). In this case eitherthe x and y coordinates of the specimen stage 18 on which the viruseshave been found or the specimen number will be stored. If no virus isfound in the field of view then the field of view will be shifted to thenext. To do this each fine tuning mechanism for x-, y-, z-axisrespectively attached to the specimen stage 18 may be used to move it tochange the field of view, or alternatively the deflective coil forcondenser system 16 may be used to transfer the position of electronbeam. Alternatively the fine adjustment mechanism may be provided at theattachment of the electron detector 17 to the electron microscope tomove the electron detector 17 itself. As can be recognized by thoseskilled in the art, the compensation for the transition and drift of theposition of specimen evidently means displacing the position of theelectron beam detector relative to the irradiation point of the electronbeam transmit through the specimen, therefore the most suitable solutionmay be chosen in accordance with the context. There are also a pluralityof solutions for the focusing compensation. In the preferred embodimentas have been described just above, the compensation for focusing may becarried out by adjusting the current of the objective lens to change thefocal distance, however, the compensation alternatively may be performedby detecting the amount of displacement D to finely adjust accordinglythe position of specimen by means of the specimen stage 18 such as inthe direction of incident axis of electron beam in case that thespecimen has been located at the focal position. This alternativesolution is just like that as shown in the flow in FIG. 20 the specimenstage is transferred into the direction of z axis after calculating theamount of defocus F. In case of a drifted specimen either the specimenstage 18 may be moved, or the mounted position of the electron detector17 may be fine-tuned in correspondence with the amount of displacement Din the plane perpendicular to the incident direction of the electronbeam.

Next, referring to FIG. 21 an example carrying out the preferredembodiment in accordance with the present invention will be describedbelow in greater details. An image (plan view) of an exemplary memorycell, observed from above thereof, transmit from the electron detector17 is as shown in the figure. In most cases the semiconductor chip isconsisted of a number of regular iterative arrays of a given pattern ofshape as shown. A contrast anomaly caused by for example a defect orcontamination debris may be included in part thereof. In FIG. 21, thereare shown defects like line segments and a round contaminant. At firstthe focusing may be adjusted as have been described above, and then thefield will be compared with a registered pattern. An example ofcomparison scheme will be described with reference to FIG. 22. By makinguse of regular pattern in the arrays to be inspected, the field of viewwill be clipped to the size of an elementary pattern. In this situationthe same size of image will be suitable for the registered image to becompared with. Similar to the viral inspection as above, the consistencyof the pattern will be evaluated, and if the correlation value fallsbelow a predetermined lower threshold the corresponding address ofmemory will be recorded. Thereafter the position of clipped field ofview will be shifted to the next to iteratively evaluate the consistencyof the pattern. Once the inspection of the entire image captured hasbeen completed, the field of view will be changed by means of forexample the specimen stage 18 or the deflective coil for condensersystem 16 and the focus will be readjusted to resume the inspection. Inthe foregoing description there are a plurality of patterns of memorycells in the image captured by the electron beam detector at thebeginning, however, it may be possible that the defects to be detectedmay be much smaller, or the contrast of the image may be much lower. Insuch a case it may be required to increase the magnification rate to alevel sufficient to the observation. To do this an image of just onememory cell will be taken with a higher magnification rate to compareone by one with the registered image without clipping. In FIG. 22 thefield of view moves from left hand side to the right hand side on thedrawing sheet. However, any other sequences are equally allowed, andsome examples are shown in FIG. 23.

The sequence should be chosen in accordance with the performance of thefine adjuster of the specimen stage and the precision of deflection ofthe deflective coil used.

In this specification some embodiments by means of a transmissionelectron microscopy (TEM) have been described by way of examples,however, the technology disclosed in the present invention may beequally applied to any other type of inspection apparatus for viewingimages by using charged particle beams such as electrons and ions,including electron microscopes, such as scanning electron microscopes(SEM), scanning transmission electron microscopes (STEM), scanning ionmicroscopes (SIM).

[Forth Embodiment]

In an apparatus for observing or inspecting a specimen by continuouslymoving the specimen stage, the transfer of specimen stage at apredetermined constant speed may become difficult if the transfer speedis faster than 5 m/sec due to the error caused by the vibration,inconstant speed, and the precision of transfer rails. This problem canbe solved by the application of the present invention, by using a firstcharged particle beam as the probe to probe the specimen to detect asecond charged particle beam emitted from the specimen to compute theerror of the current position of the specimen stage with the targetposition thereof by using the phase limitation from a plurality ofimages thus obtained to feed back the result to the specimen stage or tothe deflector which deflects the probe before the next image of thespecimen to be inspected in next turn will be captured. This allowsonset of erroneous judgments in the inspection of continuously movingspecimen to be decreased.

More specifically, The probe, first charged particle beam will becollimated to scan a predetermined area on the specimen through thedeflector and objective lens, then the second charged particle beamemitted from the specimen will be detected by the detector, and thedetected signals will be converted from analog to digital domain tostore in a storing means. The starting point of recording will be heldat a given constant position for every sessions and the scanning will bestarted over again at timing management signals or signal from thespecimen stage or the marking on the specimen. At a predetermined periodof time after the capture of first image, a second image will becaptured. These first and second images will be subjected to the Fouriertransform to determine the phase variance therebetween and will besubjected then to the invert Fourier transform to determine thedisplacement from the origin based on the distance of address in thestorage means to feed the result back to the controller of the specimenstage or to the deflector. The error found in comparison of the firstimage with the second image, due to the malfunction or incorrectoperation during transfer of the specimen stage will be decreased.

[Fifth Embodiment]

In the first embodiment described above, an example using a CCD cameraas the imaging device has been disclosed. The fundamental configurationof the CCD camera used is comprised of a scintillator 71, a photocoupler 72, and a CCD camera 73, as have been described above withreference to FIG. 6. The images formed on the scintillator 71 will befocused on the CCD camera 73 at a constant magnification rate ofprojection. In this embodiment, an example using a zoom lens for thephoto coupler 72 to variably set the magnification rate of projection incompliance with the analytic result of the displacement will bedescribed below, with reference to FIG. 27. At the bottom part of theelectron microscope is mounted a electron detector 17 in a mannersimilar to FIG. 19. The detailed configuration is shown in the figure.The electron image made of electron beam will be converted to opticalimage by the scintillator 101 placed in a vacuum. The scintillator 101is adhered on a glass substrate 103, is polished to the most suitablethickness, for example approximately 50 to 120 microns in combinationwith the accelerated electron beam at 100 kV to 400 kV. The opticalimage formed by the scintillator 101 will be focused on the imagingdevice 106 through the optical lens 105. The optical lens 105 andimaging device 106, which are devices of precision structure, arepreferably placed and operated in the atmosphere. In other words onlythe scintillator 101 is installed in the vacuum by using a vacuum seal102, the optical image will be picked up to the atmosphere through theglass substrate 103 which separates the vacuum and the atmosphere. Forthe imaging device 106 a vast majority of two dimensional detectorsincluding not only the CCD device but also any imaging devices such ascamera tubes. The procedure used for determining the amount of defocus Fcorresponding to the displacement D between the first and second imagesis identical to the first embodiment described above. In general, thesmaller the amount of defocus F is, the smaller the displacement Dbetween two images. Thus in order to find the amount of displacement Dat a higher precision, an effective way is to increase the magnificationrate of imaging when the displacement D decreased to less than a givenlimit to enlarge the displacement. To increase the magnification rate ofimaging, it will be sufficient to increase the magnification rate of theelectron microscope. However, there may often be arisen undesirableeffects, such as the change of the field of view, change in the imagecontrast resulting from the change of conditions of electron beamoptics. Therefore the inventor have devised a method for changing themagnification rate of imaging without touching the electron microscopy,by changing the magnification of the zoom of the optical lens 105. Anyzoom lens 105 commercially available in the market equipped withmotor-driven zooming mechanism may be used for the optical lens 105. Asshown in the box at the right hand bottom corner of the drawings, themagnification of zooming of the optical lens 105 may be increased to forexample 1.5 fold when the peak indicating the displacement D isapproaching to the origin. If two images at this condition are takenagain, a displacement D′ will be magnified to 1.5 fold accordingly. Ascan be seen from the foregoing description the compensation of focusingas well as drifting may be enabled at higher precision by feeding theresult of analytic images back to the election detector 17.

[Sixth Embodiment]

In the first preferred embodiment above a focus compensation using theparallax has been descried. A stigmatizer, compensator for astigmaticaberration using the parallax may also be achieved. An astigmatism is aphenomenon that, as shown in FIG. 28, the focus is distributed to anoval around the optical axis because the electromagnetic field generatedby the objective lens 14 has an oval distribution around the opticalaxis (z-axis). In other words the focus has a distribution described byF (β)=F+A cos² (β−βA), depending on the azimuth β. In this equation Fdesignates to the mean of F (β), referred to as the amount of defocus ingeneral the A designates to the amount of astigmatism, and β A to theastigmatic orientation.

The apparatus and the process flow depicted in FIG. 29 may be used tofind the focus distribution around the optical axis by the focusanalysis using the parallax to analyze the amount and orientation ofastigmatism to feed the result back to the stigmatizer 141. A first TEMimage will be taken by irradiating the specimen with the incidentelectron beam from a first direction approximately in parallel to theoptical axis, the z-axis, and a second TEM image will be taken byirradiating the specimen with the incident electron beam from a seconddirection tilted by an angle α from the z-axis. The azimuth of thesecond direction with respect to the x-axis will be β₂. The displacementD (β₂) between the first TEM image and the second TEM image will beanalyzed by the displacement analysis processor using phase variance ofFourier transform images 20, which will in turn send thus founddisplacement D (β₂) to the computer 19, which further will computes theamount of defocus F (β₂) at the azimuth β₂. Thereafter, a third TEMimage will be taken by irradiating the specimen with the electron beamfrom a third direction where the direction is tilted by angle α from thez-axis, and the azimuth to the x-axis becomes β₃, in order to analyzethe displacement D (β₃) between the first TEM image and the third TEMimage to determine the amount of defocus F (β₃) at the azimuth β₃ to thex-axis. Then the same procedure of analysis will be applied to aplurality of azimuths β_(n) to determine the distribution of azimuth atthe focal point.

Any one of fitting methods including such as least-square method will beused to identify the amount of defocus, astigmatism, and the astigmaticorientation from the focal point F (β_(n)) at each orientation. Withrespect to the astigmatic orientation there may be a case in which theazimuth of incident electron beam may not be in parallel to thedirection of displacement vector, by the influence of image rotationbeing generated within the electron lenses. Since the difference betweenthe orientation of incident electron beam and the direction ofdisplacement vector may be determined once conditions of lens such asthe magnification rate has been decided, the difference at each of lensconditions may be stored in the computer to correct the astigmaticorientation based on the stored difference.

Based on the result of the astigmatism analysis, the current values I sxand I sy of the stigmatizer 141, required in order for the amount ofastigmatism A to become zero, will be computed so as to adjust thestigmatizer 141 through the stigmatizer control circuit 141′.

The astigmatic analysis needs a precision by two digits or more superiorto the precision of defocus analysis. In a conventional focus analysissystem using the cross-correlation for the analysis of displacement itwill be very difficult to satisfy a precision required to carry out thestigmation. The analysis of astigmatism is an analysis of focaldistribution, so that a large number of times of displacement analysiswill be required. The system disclosed herein, equipped with thedisplacement analysis processor using phase variance of Fouriertransform images 20, based on a digital signal processor (DSP), mayperform one focusing analysis within a second, allowing one analysis ofastigmatism to be completed within a few seconds.

The performance of apparatus compensating for an electron microscopebased on the displacement between the images taken by the electronmicroscope such as the focusing analysis using the parallax is largelydepending on the displacement analysis. In the analysis method ofdisplacement used in the conventional compensator systems the precisionof analysis in theory cannot be smaller than the size of a pixel of theelectron detector 17. However, in accordance with the present invention,a precision of analysis smaller than the size of a pixel may beobtained. The apparatus in accordance with the present invention iscapable to adjust the focusing at a precision as fine as a skillfuloperator. Although the time required for analysis and the cost ofhardware will be increased when improving the performance in an attemptto improve the precision of focal analysis, such as subdividing theimage to be inspected into still smaller pieces, the present invention,which may improve the precision of the analysis of displacement byaltering the analysis method of displacement, allows the precision offocusing analysis to be improved without additional time of analysis andcost of hardware.

Furthermore, in accordance with the present invention, the consistencybetween paired images is indicated as a correlation. The operator of theelectron microscope may check the reliability of the analytic resultoutput. Incorrect operation may be prevented by setting a lowerthreshold of correlation values so as not to limit the adjustment of thelens system when a calculated correlation value is less than the lowerthreshold. In an automatic inspection apparatus, the operator may checkto see later whether or not the automatic compensation has beenperformed correctly, by storing the correlation values in the focalanalysis and the results of focal analysis, allowing unmanned operationto be performed.

The analysis of displacement in accordance with the present invention isa method making use of the phase component of images, which is almostimmune to the variance of background, and is operable if there is asufficient common area of paired images, even when some extent of shadowof aperture covers the images. The system in accordance with the presentinvention is still operable when the TEM is not sufficiently configured.In brief, an operator unfamiliar with the operation of TEM may use thesystem.

The foregoing description of some preferred embodiments of the inventionhas been presented for purposes of illustration and description. It isnot intended to be exhaustive or to limit the invention to the preciseforms disclosed, and modifications and variations are possible in lightof the above teachings or may be acquired from practice of theinvention. The embodiments are chosen and described in order to explainthe principles of the invention and its practical application to enableone skilled in the art to utilize the invention in various embodimentsand with various modifications as are suited to the particular usecontemplated it is intended that the scope of the invention be definedby the claims appended hereto, and their equivalents.

1. An electron beam apparatus, comprising: a source of charged particle beam; a specimen stage for holding a specimen; a specimen stage controller for moving the specimen stage; a deflector coil for deflecting an incident position of a first charged particle beam into the specimen; a detector for detecting a second charged particle beam from the specimen and generating an image signal; a computer for obtaining a plurality of images from the image signal to calculate a transfer speed of the specimen; and a deflector coil controller for controlling the operation of the deflector coil so as to cancel out the influence of transfer of the specimen stage.
 2. An electron beam apparatus according to claim 1, wherein: the specimen stage controller moves the specimen stage at a certain velocity; and the deflector coil controller controls the deflector coil to shift the second charged particle beam at a substantially same velocity as the specimen stage.
 3. An electron beam apparatus according to claim 1, wherein: the specimen stage controller stops the specimen stage; and the deflector coil controller controls the deflector coil to shift the second charged particle beam so as to cancel out a specimen drift.
 4. A method of operating an electron microscope, comprising the steps of: placing a specimen on a specimen stage; continuously moving the specimen stage; irradiating the specimen with a first electron beam; taking an image by a second electron beam from the specimen; capturing a plurality of taken images for storage; performing an image processing on said plurality of taken images to determine a consistency between the plurality of taken images; determining an amount of displacement between the plurality of taken images; and controlling a deflector coil to deflect the irradiation of the first electron beam to the specimen so as to cancel out the transfer of the specimen stage based on the determined consistency and displacement. 